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説明:

Deciphering metabolic enzyme catalysis in living cells remains a formidable challenge due to the limitations of in vivo assays, which focus on enzymes isolated from respiration. This study introduces an innovative whole-cell electrochemical assay to reveal the Michaelis–Menten landscape of respiratory enzymes amid complex molecular interactions. We controlled the microbial current generation’s rate-limiting step, extracting in vivo kinetic parameters (Km, Ki, and kcat) for the periplasmic nitrite (NrfA) and fumarate (FccA) reductases. Notably, while NrfA kinetics mirrored those of its purified form, FccA exhibited unique kinetic behavior. Further exploration using a mutant strain lacking CymA, a periplasmic hub protein, revealed its crucial role in modulating FccA’s kinetics, challenging the prevailing view that molecular crowding is the main cause of discrepancies between in vivo and in vitro enzyme kinetics.

権利情報:

• Creative Commons BY-NC-ND Attribution-NonCommercial-NoDerivs 4.0 International
  

キーワード: Michaelis–Menten equation, interprotein interaction, enzymatic activity

刊行年月日: 2025-03-25

出版者: Proceedings of the National Academy of Sciences

掲載誌:

• Proceedings of the National Academy of Sciences (ISSN: 00278424) vol. 122 issue. 12 e2418926122

研究助成金:

• MEXT | JST | ACT-X JPMJAX211C
• MEXT | Japan Society for the Promotion of Science 20K15428
• MEXT | Japan Science and Technology Agency JPMJGX23B2
• MEXT ARIM JPMXP1223NM5212
• University of Tsukuba None
• MEXT | JST | Fusion Oriented REsearch for disruptive Science and Technology JST FOREST
• MEXT | Japan Society for the Promotion of Science 22KK0242
• MEXT | Japan Society for the Promotion of Science 23K23532

原稿種別: 出版者版 (Version of record)

MDR DOI:

公開URL: https://doi.org/10.1073/pnas.2418926122

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更新時刻: 2025-12-24 16:46:17 +0900

MDRでの公開時刻: 2025-12-25 08:19:49 +0900